<2022-06-21 Tue> David biweekly meeting

project updates

Project 4: Reconstitution of endocytic actin network

recent progress

  • Danielle had to delay sectioning samples due to covid in her family; should be resuming this week

next steps

  • EM imaging when sectioning/staining is done

Project 5: Experimental role of phase-separated protein droplet in endocytosis

recent progress

  • eps15 appears to express well in e. coli in test expression (replicating Stachowiak lab paper)

    2022-06-20_23-17-19_5.11.1I3.png
    • green ladder band is ~100kD, same as eps15
    • lanes: 0,2,4,6,20,22,24,26,28 hr induction

next steps

  • see plan for Nikhil's protein purification project
  • brainstorming on how to adapt this system for complementing the model

    2022-04-26_09-32-29_screenshot.png
    • Tyler suggested finding a substrate with holes approximately the size of the nanoparticles in order to achieve lower curvature
  • try making cytoplasmic extract from ede1 overexpression strain
    • already tried the 3x adaptor deletion strain; did not produce condensates

Project 7: Mechanical regulation of CME by protein LLPS

recent progress

  • with Tyler's help, identified a term that was missing from my model that caused the simulations I showed last time to not run correctly

next steps

  • add in the missing term
  • run a simulation of successful endocytosis mediated by the coat bending energy
  • build final version of model with protein condensate

plan for Nikhil's protein purification project

  1. express and purify His-tagged GFP in E. coli
  2. insert GFP into His6-eps15 plasmid from Stachowiak lab
  3. express and purify His6-eps15-GFP from E. coli
  4. purify His6-eps15
  5. purify Fcho1
  6. reproduce phase separation experiments

Date: \today

Author: Max Ferrin

Email: ferrinm@berkeley.edu

Created: 2022-06-20 Mon 23:39

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